Previously, I purified the HTT N-HEAT_81-1643 domain and showed that the HTT N-HEAT_81-1643 domain elutes early from a Superose 6 column 10/300 (https://zenodo.org/record/3462496#.XjsVXSNOk2w). Initial biophysical characterization by DLS showed samples of the HTT N-HEAT_81-1643 construct are made up of large particles ( https://zenodo.org/record/3562523#.Xjw57iN, https://zenodo.org/record/3561096#.Xjw6ByNOk2w). Thus, our initial results all pointed out at having high oligomeric states of the HTT N-HEAT_81-1643 construct in solution. To rule out the possibility of a weak interaction with nucleic acid material which could cause the large particle size in solution, we tested whether we could remove the nucleic acid material by adding an additional purification step with heparin resin. However, purification using the additional heparin step showed no improvement in the purity of the sample from other protein impurities or nucleic acid material (https://zenodo.org/record/3562523#.XjsVyyNOk2w). Determination of ideal buffer conditions for the HTT N-HEAT_81-1643 construct using DSLS and DSF (https://zenodo.org/record/3562523#.Xjw57iN https://zenodo.org/record/3519364#.Xjw6ICNOk2w, https://zenodo.org/record/3561087#.Xjw6LiNOk2w) was not possible as no significant changes in the Taag or Tm were observed for the conditions tested. Further, the data for both DSF and DSLS could have presented challenges to fit (e.g. The DSF data showed very high initial fluorescence while the DSLS data showed the Tagg ­ for this construct was at the end of the detection limit ~ 90 °C). Thus, to further assess if we could find an ideal buffer condition where the HTT N-HEAT_81-1643 construct is the most stable and monomeric, we tested different purification conditions.

Dr. Harding is the recipient of the Huntington's Disease Society of America Berman Topper Career Development Fellowship which funds and supports this research, in addition to generous funding from the CHDI Foundation and the Huntington Society of Canada. The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada through Ontario Genomics Institute [OGI-055], Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck KGaA, Darmstadt, Germany, MSD, Novartis Pharma AG, Ontario Ministry of Research, Innovation and Science (MRIS), Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome.